U.S. patent application number 15/468295 was filed with the patent office on 2017-07-06 for pharmaceutical composition comprising rimeporide for treating diseases associated with insulin resistance and beta-cell dysfunction.
This patent application is currently assigned to Merck Patent GmbH. The applicant listed for this patent is Merck Patent GmbH. Invention is credited to Norbert BEIER, Ulrich BETZ, Marian BRAENDLE, Wolfgang SCHOLZ.
Application Number | 20170189354 15/468295 |
Document ID | / |
Family ID | 40775400 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170189354 |
Kind Code |
A1 |
BEIER; Norbert ; et
al. |
July 6, 2017 |
PHARMACEUTICAL COMPOSITION COMPRISING RIMEPORIDE FOR TREATING
DISEASES ASSOCIATED WITH INSULIN RESISTANCE AND BETA-CELL
DYSFUNCTION
Abstract
The invention relates to a pharmaceutical composition comprising
as active ingredient an effective amount of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, or derivatives
thereof, for the prophylaxis and therapy of Type II diabetes
mellitus, the Metabolic syndrome, diabetic nephropathy and/or
neuropathy. Another object of the invention concerns the use of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, or derivatives
thereof, for the enhancement of insulin sensitivity and the
preservation or increase of .beta.-cell compensation.
Inventors: |
BEIER; Norbert; (Reinheim,
DE) ; SCHOLZ; Wolfgang; (Eschborn, DE) ; BETZ;
Ulrich; (Reinheim, DE) ; BRAENDLE; Marian;
(Starzach, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Patent GmbH |
Darmstadt |
|
DE |
|
|
Assignee: |
Merck Patent GmbH
Darmstadt
DE
|
Family ID: |
40775400 |
Appl. No.: |
15/468295 |
Filed: |
March 24, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12991524 |
Nov 8, 2010 |
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PCT/EP2009/002701 |
Apr 11, 2009 |
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15468295 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/155 20130101;
A61P 3/04 20180101; A61K 9/0053 20130101; A61P 13/12 20180101; A61P
25/02 20180101; A61P 3/10 20180101 |
International
Class: |
A61K 31/155 20060101
A61K031/155; A61K 9/00 20060101 A61K009/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 9, 2008 |
EP |
08008763.8 |
Claims
1.-13. (canceled)
14. A method for the treatment of the arising symptoms of a disease
that is associated with .beta.-cell dysfunction having reduced
.beta.-cell compensation, comprising administering to a patient an
effective amount of an active ingredient which is
2-methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, and/or a
physiologically acceptable salt and/or solvate thereof, such that
the .beta.-cell dysfunction in the patient is decreased, wherein
the 2-methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine and/or a
physiologically acceptable salt and/or solvate thereof acts as
direct insulin secretagogue.
15. The method of claim 14, wherein the method is for treating the
arising symptoms of type II diabetes mellitus associated with
.beta.-cell dysfunction and reduced .beta.-cell compensation.
16. The method of claim 14, wherein the active ingredient is a
2-methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine salt selected
from the hydrochloride, methanesulfonate, hemi-sulfate,
hemi-fumerate and hemi-malate salts.
17. The method of claim 14, wherein the active ingredient is
2-methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine hydrochloride
hydrate.
18. The method of claim 14, wherein the active ingredient is
administered by oral or parenteral administration.
19. The method of claim 14, wherein the active ingredient is
administered by oral administration.
20. A method according to claim 14, wherein the pancreatic
.beta.-cell compensation in the patient is preserved to at least
70% of baseline prior to usage.
21. A method according to claim 20, wherein the active ingredient
is administered for a period of at least 4 weeks.
22. A method for the treatment of .beta.-cell dysfunction having
reduced .beta.-cell compensation in a patient, comprising
administering to the patient an effective amount of an active
ingredient which is
2-methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, and/or a
physiologically acceptable salt and/or solvate thereof, wherein the
2-methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine and/or a
physiologically acceptable salt and/or solvate thereof acts as
direct insulin secretagogue.
23. A method according to claim 14, wherein the active ingredient
maintains or increases .beta.-cell response in the patient.
24. A method according to claim 14, wherein the active ingredient
is administered to the patient in a dose of 1 to 600 mg per dose
unit.
25. A method according to claim 14, wherein the active ingredient
is administered to the patient in a dose of 5 to 100 mg per dose
unit.
26. A method according to claim 14, wherein the active ingredient
is administered to the patient in a daily dose of between 0.02 and
200 mg/kg of patient body weight.
27. A method according to claim 14, wherein the active ingredient
is administered to the patient in a daily dose of between 20 and
100 mg/kg of patient body weight.
Description
[0001] The invention relates to a pharmaceutical composition
comprising as active ingredient an effective amount of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, or derivatives
thereof, for the prophylaxis and therapy of Type II diabetes
mellitus, the Metabolic syndrome, diabetic nephropathy and/or
neuropathy. Another object of the invention concerns the use of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, or derivatives
thereof, for the enhancement of insulin sensitivity and the
preservation or increase of .beta.-cell compensation.
[0002] Metabolic diseases such as obesity, insulin resistance (IR)
and dyslipidemia are emerging as dominant causes of morbidity and
mortality worldwide. Especially over the last decades, IR has
become a highly prevalent condition in the general public, with
enormous consequences for the public health system. IR is defined
as the reduced, non-adequate response of the body to the normal
actions of insulin. IR is an important risk factor for the
development of cardiovascular disease and Type II diabetes mellitus
(T2DM). In addition, IR is associated with a variety of
cardiovascular risk-factors (obesity, dyslipidemia, hypertension
and blood clotting disturbances) that when exhibited collectively
is referred to as the Metabolic syndrome or Syndrome X.
Considerable evidence now exists that IR may be the unifying causal
factor underlying the Metabolic Syndrome (Turner & Hellerstein
(2005) Curr Opin Drug Discovery & Develop 8(1): 115-126).
[0003] Current therapeutic interventions aiming to directly improve
the insulin responsiveness of the tissues apply thiazolidinediones
(TZDs). However, while the TZDs have been shown to improve
whole-body insulin sensitivity, they recently have become known to
increase the risk of heart failure and cardiovascular
complications. Therefore, alternatives for the treatment of IR are
necessary in the fight against the growing epidemic of deranged
metabolic diseases, with one of its features being IR.
[0004] In addition to IR, pancreatic .beta.-cell dysfunction plays
a pivotal role in the progression from the pre-diabetic to the
diabetic state. The recent development of agents, such as exendin-4
(Xu et al. Diabetes (1999) 48(12): 2270-2276; DeFronzo et al.
(2005) Diabetes Care 28(5): 1092-1100) or a sitagliptin analog (Mu
et al. (2006) Diabetes 55(6): 1695-1704), that may stimulate
.beta.-cell regeneration and increase .beta.-cell mass has focused
further interest on .beta.-cell mass as a therapeutic target in
T2DM.
[0005] Therefore, the technical problem forming the basis of the
present invention is to provide a pharmaceutical composition
allowing an effective application in the prevention or therapy of
diseases that are associated with insulin resistance and/or
.beta.-cell dysfunction, especially such compositions that improve
the therapeutic efficacy and minimize adverse effects.
[0006] The present invention solves this problem by providing a
pharmaceutical composition comprising as active ingredient an
effective amount of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, and/or a
physiologically acceptable salt and/or solvate thereof, for the
prophylactic or therapeutic treatment and/or monitoring of diseases
that are associated with insulin resistance and/or .beta.-cell
dysfunction.
[0007] It has been surprisingly demonstrated by the inventors that
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine and said
derivatives can be applied as active ingredients in a
pharmaceutical composition in order to tackle medical indications
such as T2DM and diseases being linked thereof.
[0008] Before filing this application, it is only known from EP 0
758 644 B1 that sulfonylbenzoyl-guanidines are inhibitors of the
cellular Na.sup.+/H.sup.+ antiporter, i.e. they inhibit the
Na.sup.+/H.sup.+ exchange mechanism of the cells, thereby being
good antiarrhythmic agents, which are suitable, in particular, for
the treatment of arrhythmia occurring as a consequence of oxygen
deficiency. The substances exhibit a good cardioprotective action
and are therefore suitable for the treatment of acute myocardial
infarction, infarction prophylaxis, post-infarction treatment,
chronic cardiac insufficiency and for the treatment of angina
pectoris. They furthermore counter all pathological hypoxic and
ischemic damage, enabling the illnesses caused primarily or
secondarily thereby to be treated. Owing to the protective action
of these substances in pathological hypoxic or ischemic situations,
further applications arise in surgical interventions for protection
of organs with temporarily reduced supply, in organ transplants for
protection of the removed organs, in angioplastic vascular or
cardiac interventions, in ischemia of the nervous system, in the
therapy of shock states and for the prevention of essential
hypertonia. In addition, the compounds are suitable for diagnostic
use for the recognition of illnesses accompanied by increased
activity of the Na.sup.+/H.sup.+ antiporter, for example in
erythrocytes, thrombocytes or leukocytes. Prior art has
additionally suggested to employ these substances as therapeutic
agents in illnesses caused by cell proliferation, such as
arteriosclerosis, diabetes and late complications of diabetes,
tumor illnesses, fibrotic illnesses, in particular of the lungs,
liver and kidneys, and organ hypertrophia and hyperplasia.
[0009] But now, the present invention reveals the increase in
whole-body IR while .beta.-cell compensation is simultaneously
preserved. These phenomena are stimulated by the impact of the
compound 2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, which
forms the basis of the inventive remedy for such specified clinical
pictures as T2DM and the Metabolic syndrome.
[0010] The aforementioned compound exhibits very valuable
pharmacological properties along with good tolerability. Weight
gain, fasting or random blood glucose levels are not changed at any
dose tested in clinical trials. In contrast, fasting insulin is
decreased in comparison to the pre-treatment by
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine. The insulin
area-under-the-curve (AUC) value is also decreased after an oral
glucose load while the AUC response to the glucose load remains
similarly at pre- and post-treatment. It is an unexpected finding,
however, that the lower insulin levels do not arise from the
age-related drop in .beta.-cell function, since the compound of the
invention maintains or even increases .beta.-cell response.
Accordingly, 2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine
significantly enhances the peripheral insulin sensitivity.
[0011] Said biological activities of the compound of the invention
may be determined by techniques known to the skilled artisan.
Suitable experimental animals are, for example, mice, rats,
guinea-pigs, dogs, cats, apes or pigs. The gold standard for the
in-vivo assessment of IR is the euglycemic-hyperinsulinemic glucose
clamp. Other tools are the steady-state plasma glucose (SSPG) test
or the frequently sampled intravenous glucose tolerance test.
Available techniques for measuring pancreatic .beta.-cell
proliferation in-vivo include the [.sup.3H]thymidine (.sup.3HdT) or
5-bromodeoxyuridine (BrdU) approaches. Techniques that are suitable
to determine changes in insulin sensitivity and .beta.-cell
dynamics have to be sensitive, reproducible, operationally simple
and relatively high-throughput.
[0012] As stated before, insulin resistance is an important risk
factor for the development of type 2 diabetes mellitus and
cardiovascular disease. The pathogenesis of type 2 diabetes
involves not only insulin resistance, but also progressive
pancreatic insufficiency. A reliable, easily performed quantitative
test has been developed. By means of this deuterated-glucose
disposal test (.sup.2H-GDT) both dimensions of type 2 diabetes can
be quantified.
[0013] The glycolytic disposal of a glucose load by peripheral
tissues, such as skeletal muscle, depends upon a number of
insulin-dependent steps, including the transport, phosphorylation
and passage through enzymes of the glycolytic pathway. In addition,
glucose effectiveness, actions of glucoses per se to both inhibit
hepatic glucose production and accelerate its uptake into tissues,
also contributes to disposal of an oral glucose load. The
.sup.2H-GDT measures the rate of uptake, phosphorylation and
glycolytic metabolism of glucose and thus can be used to quantify
both insulin resistance (IR) and the adequacy of pancreatic
.beta.-cell compensation. The .sup.2H-GDT consists of an oral
deuterated-glucose challenge followed by the measurement of heavy
water (.sup.2H.sub.2O) production and plasma insulin
concentrations. Because H atoms are released into tissue water
during the glycolytic metabolism of glucose, measurement of
.sup.2H.sub.2O production represents the rate of whole-body
glycolytic disposal of glucose. .sup.2H.sub.2O production corrected
for ambient insulin concentrations (i.e. glycolytic metabolism per
unit of insulin) reveals tissue insulin sensitivity. Pancreatic
compensation when corrected for the glycemic excursion (to account
for the contribution of glucose effectiveness), reveals the degree
to which glycolytic metabolism (absolute .sup.2H.sub.2O production)
is matched to insulin sensitivity. In insulin resistant states, the
pancreatic compensation is incomplete and glucose tolerance is
impaired.
[0014] In detail, the recently developed, deuterated glucose
disposal test (.sup.2H-DGT) involving stable isotope-mass
spectrometric assessment of whole-body glycolysis allows the
assessment of IR by measuring the .sup.2H.sub.2O-production per
unit of plasma insulin*glucose, which is based on the rate of
release of deuterium (.sup.2H) from an (oral) load of the animal or
individual with deuterated [6,6'-.sup.2H.sub.2]glucose and the
determined plasma insulin concentrations (Turner & Hellerstein
(2005) Curr Opin Drug Discovery & Develop 8(1): 115-126). In
addition, the degree of pancreatic .beta.-cell compensation to IR
can be assessed by measuring the absolute .sup.2H.sub.2O production
achieved after the [6,6'-.sup.2H.sub.2]glucose load. Adequacy of
pancreatic compensation can be assessed by distinguishing between
the glycolytic disposal per unit of ambient insulin (reflecting
insulin sensitivity) and the absolute rate of glucose utilization
achieved (reflecting pancreatic compensation to IR).
[0015] The .sup.2H-GDT is designed to adhere to the following
principles: i) ambient glucose and insulin concentrations should
reflect metabolic conditions physiologically relevant, ii) the test
should measure insulin-mediated glucose utilization by tissues and
reveal IR in established models, and iii) the method should reflect
comparable metabolic conditions as other tests of IR that are
proven to be predictive for cardiovascular outcomes and T2DM risk.
Serum insulin concentrations in the "dynamic range" between basal
and maximal glucose utilization conditions fulfill these criteria
(Beysen et al. (2007) Diab Care 30:1143-1149). Furthermore, the
.sup.2H-GDT, which measures the whole-body glycolysis in animals or
humans in a quantitative manner, strongly correlates with the
euglycemic-hyperinsulinemic glucose clamp or SSPG tests. The
utilization of said kinetic assay is consequently preferred in the
scope of the invention in order to determine the in-vivo effect of
an agent on insulin sensitivity as well as insulin compensatory
responses, in particular at relatively high-throughput, and in many
commonly used preclinical animal models. In addition, the
.sup.2H-GDT is completely translational into the clinical setting
with a similar degree of simplicity and throughput.
[0016] The active ingredient of the pharmaceutical composition
according to the invention is an effective amount of the compound
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, or
pharmaceutically usable derivatives thereof. Such derivatives can
be solvates of the matching compounds, salts or pro-drugs, for
instance. Preference is given to solvates and/or physiologically
acceptable salts.
[0017] A "pharmaceutical composition" in the meaning of the
invention is any agent in the field of medicine, which comprises
one or more substances or preparations thereof and can be used in
prophylaxis, therapy, follow-up or aftercare of patients who suffer
from diseases, which are associated with insulin resistance and/or
.beta.-cell dysfunction, in such a way that a pathogenic
modification of their overall condition or of the condition of
particular regions of the organism could establish at least
temporarily.
[0018] The term ".beta.-cell dysfunction" relates to any
malfunction in proliferation of .beta.-cells and/or their cellular
metabolism, which results in a diminished viability and/or
metabolic activity with the consequence of reduced .beta.-cell
compensation and insulin levels. Such loss of .beta.-cell function
may be e.g. either caused by age-related deterioration or developed
in the progression to type II diabetes, but any other causes shall
be not excluded. A clear linkage of .beta.-cell dysfunction and
insulin resistance is preferred in the scope of the invention, so
that the inventive pharmaceutical composition targets diseases
associated with both, .beta.-cell dysfunction and insulin
resistance, in particular.
[0019] The terms "effective amount" or "effective dose" or "dose"
are interchangeably used herein and denote an amount of the
pharmaceutical compound having a prophylactically or
therapeutically relevant effect on a disease or pathological
conditions. A prophylactic effect prevents the outbreak of a
disease. A therapeutically relevant effect relieves to some extent
one or more symptoms of a disease or returns to normality either
partially or completely one or more physiological or biochemical
parameters associated with or causative of the disease or
pathological conditions. The respective dose or dosage range for
administering the pharmaceutical composition according to the
invention is sufficiently high in order to achieve the desired
prophylactic or therapeutic effect of reducing symptoms of the
aforementioned diseases, particularly T2DM and said other upcoming
diseases. It will be understood that the specific dose level,
frequency and period of administration to any particular human will
depend upon a variety of factors including the activity of the
specific compound employed, the age, body weight, general state of
health, gender, diet, time and route of administration, rate of
excretion, drug combination and the severity of the particular
disease to which the specific therapy is applied. Using well-known
means and methods, the exact dose can be determined by one of skill
in the art as a matter of routine experimentation.
[0020] The compound
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, which is used
in the inventive manner, and the starting materials for its
preparation, respectively, are produced by methods known per se, as
described in the literature (for example in standard works, such as
Houben-Weyl, Methoden der organischen Chemie [Methods of Organic
Chemistry], Georg-Thieme-Verlag, Stuttgart), i.e. under reaction
conditions that are known and suitable for said reactions. Use can
also be made of variants that are known per se, but are not
mentioned in greater detail herein. If desired, the starting
materials can also be formed in-situ by leaving them in the
un-isolated status in the crude reaction mixture, but immediately
converting them further into the compound according to the
invention. On the other hand, it is possible to carry out the
reaction stepwise.
[0021] For example, a process for the preparation of
alkylbenzoylguanidine derivatives is described in EP 0 758 644 B1.
In addition, EP 1 282 598 B1 teaches a method for the production of
sulfonylbenzoylguanidium salts, wherein the preparation is
particularly preferred in respect of the compound
N-(4,5-bis-methanesulfonyl-2-methylbenzoyl)guanidine hydrochloride.
Another process for preparing
N-(4,5-bis-methanesulfonyl-2-methylbenzoyl)guanidine hydrochloride
and the hydrochloride hydrate is disclosed in DE 199 51 418 A1. The
aforementioned three documents are incorporated as reference in the
disclosure of the invention hereby.
[0022] "Solvates" are regarded as attachments of inert solvent
molecules to the compound, which are formed by respective mutual
forces of attraction. Preferably, solvates are mono hydrates,
dehydrates or alcoholates.
[0023] The provision of a salt can be performed by converting an
acid of the compound
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine into the
associated acid-addition salt using a base, for example by the
reaction of equivalent amounts of the acid and the base in an inert
solvent, such as ethanol, followed by evaporative concentration.
Particular suitable bases for this reaction are those giving
physiologically acceptable salts. For instance, an acid of the
aforementioned compound can be converted into the corresponding
metal salt, particularly an alkali metal or alkaline earth metal
salt, or into the corresponding ammonium salt, using a base, for
example sodium hydroxide, potassium hydroxide, sodium carbonate or
potassium carbonate. Suitable for this reaction are, in particular,
also organic bases, which give physiologically acceptable salts,
such as ethanolamine.
[0024] On the other hand, a base of the compound
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine can be converted
into the associated acid-addition salt using an acid, for example
by reaction of equivalent amounts of the base and the acid in an
inert solvent, such as ethanol, followed by evaporation. Suitable
acids for this reaction are, in particular, those which give
physiologically acceptable acids. For instance, it is possible to
use inorganic acids, for example sulfuric acid, nitric acid,
hydrohalic acids, such as hydrochloric acid or hydrobromic acid,
phosphoric acids, such as orthophosphoric acid, or sulfamic acid,
furthermore organic acids, in particular aliphatic, alicyclic,
araliphatic, aromatic or heterocyclic monobasic or polybasic
carboxylic, sulfonic or sulfuric acids, for example formic acid,
acetic acid, propionic acid, pivalic acid, diethyl acetic acid,
malonic acid, succinic acid, pimelic acid, fumaric acid, maleic
acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic
acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane- or
ethanesulfonic acid, ethanedisulfonic acid, 2-hydroxyethanesulfonic
acid, benzenesulfonic acid, p-toluenesulfonic acid,
naphthalenemono- and -disulfonic acids, and laurylsulfuric acid.
Salts with physiologically unacceptable acids, far example
picrates, can be used for the isolation and/or purification of the
compound 2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine.
[0025] Preferred derivatives are
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine salts selected
from the group of hydrochloride, methanesulfonate, hemi-sulfate,
hemi-fumerate and hemi-malate. In a more preferred embodiment of
the invention, 2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine
hydrochloride represents the active ingredient of the
pharmaceutical composition.
[0026] The pharmaceutical composition may also comprise mixtures of
the compound and at least a singe derivative, or mixtures of
derivatives, respectively, which may comprise solvates and/or
salts, for instance. It is most preferred to use
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine hydrochloride
hydrate.
[0027] Furthermore, the pharmaceutically usable derivatives may
include pro-drug derivatives, i.e. modified compounds having
supplemental alkyl groups, acyl groups, sugar molecules or oligo
peptides, which are immediately cleaved into the active inventive
compound 2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine within
the organism. Herein, biologically degradable polymer derivates of
the compound according to the invention are also included as e.g.
described in Int. J. Pharm. 115, 61-67 (1995). The compound of the
invention can be obtained by liberating it from their functional
derivatives by solvolysis, in particular hydrolysis, or by
hydrogenolysis.
[0028] The active ingredient according to the invention can also be
fused or complexed with another molecule that promotes the directed
transport to the destination, the incorporation and/or distribution
within the target cells.
[0029] Furthermore, the active ingredient may be administered alone
or in combination with other treatments. A synergistic effect may
be achieved by using more than one compound in the pharmaceutical
composition, i.e. the compound
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine is combined with
at least another agent as active ingredient, such as glitazone,
exenatide, pramlintide or TZDs. The compounds can be used either
simultaneously or sequentially.
[0030] The pharmaceutical composition of the invention can be
employed as medicament in human and veterinary medicine. It is
particularly preferred that the diseases, which are associated with
IR and/or .beta.-cell dysfunction, are represented by T2DM, the
Metabolic syndrome, nephropathy and/or neuropathy.
[0031] In a preferred embodiment of the invention, the disease
underlying the invention is T2DM. The medical indication "T2DM" is
a progressive disease which involves the development of IR and
other metabolic abnormalities, long before overt glucose
intolerance and fasting hyperglycemia are exhibited.
[0032] The medical indication "Metabolic syndrome" is a combination
of medical disorders that increase the risk of developing
cardiovascular diseases. In addition to central obesity, two
further symptoms and features have to be fulfilled for
classification as the Metabolic syndrome: fasting hyperglycemia
(expressed by Type II diabetes mellitus, impaired fasting glucose,
impaired glucose tolerance or insulin resistance), high blood
pressure and lipometabolic disorder (e.g. decreased HDL cholesterol
and/or elevated triglycerides).
[0033] The medical indication "nephropathy" relates to diseases of
the kidney and kidney function, which are mainly caused
non-inflammatorily. The challenging subtype in the scope of the
invention is reflected by diabetic nephropathy (nephropatia
diabetica), which is also known as Kimmelstiel-Wilson syndrome and
intercapillary glomerulonephritis. It is a progressive kidney
disease caused by angiopathy of capillaries in the kidney
glomeruli, and characterized by nephrotic syndrome and nodular
glomerulosclerosis. It is due to longstanding diabetes mellitus,
and is a prime cause for dialysis in many Western countries.
[0034] The medical indication "neuropathy" is usually short for
peripheral neuropathy. Peripheral neuropathy is defined as deranged
function and structure of peripheral motor, sensory, and autonomic
neurons, involving either the entire neuron or selected levels.
Neuropathies often arise secondarily from other diseases, such as
diabetes mellitus, or neurotoxic substances, such as alcohol
abuse.
[0035] The pharmaceutical composition of the invention is produced
in a known way using common solid or liquid carriers, diluents
and/or additives and usual adjuvants for pharmaceutical engineering
and with an appropriate dosage. The amount of excipient material
that is combined with the active ingredient to produce a single
dosage form varies depending upon the host treated and the
particular mode of administration. Suitable excipients include
organic or inorganic substances that are suitable for the different
routes of administration, such as enteral (e.g. oral), parenteral
or topical application, and which do not react with
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, or derivatives
thereof. Examples of suitable excipients are water, vegetable oils,
benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol
triacetate, gelatin, carbohydrates, such as lactose or starch,
magnesium stearate, talc, and petroleum jelly.
[0036] In the meaning of the invention, an "adjuvant" denotes every
substance that enables, intensifies or modifies a specific response
against the active ingredient of the invention if administered
simultaneously, contemporarily or sequentially. Known adjuvants for
injection solutions are, for example, aluminum compositions, such
as aluminum hydroxide or aluminum phosphate, saponins, such as
QS21, muramyldipeptide or muramyltripeptide, proteins, such as
gamma-interferon or TNF, M59, squalen or polyols.
[0037] The active ingredients of the instant composition are
adapted in forms which are suitable for oral administration, such
as tablets, film tablets, coated tablets, lozenges, capsules,
pills, powders, granules, syrups, juices, drops, solutions,
dispersions, suspensions or depot forms thereof; for transdermal
administration, such as solutions, suspensions, creams, ointments,
powders, gels, emulsions or band-aids; for parental administration,
such as suppositories, suspensions, emulsions, implants or
solutions, preferably oily or aqueous solutions; for rectal
administration, such as suppositories in particular; for topical
application, such as ointments, creams, pastes, lotions, gels,
sprays, foams, aerosols, solutions (for example solutions in
alcohols, such as ethanol or isopropanol, acetonitrile, DMF,
dimethylacetamide, 1,2-propanediol or mixtures thereof with one
other and/or with water) or powders; and for intravenous infusion,
subcutaneous injection or intramuscular administration, examples
for the latter three are solutions and suspensions. The active
ingredients can also be adapted for transmucosal, transurethal,
vaginal or pulmonary administration in the appropriate formulations
given above.
[0038] In a preferred embodiment of the present invention, the
pharmaceutical composition is orally or parenterally administered,
more preferably orally. In particular, the active ingredient is
provided in a water-soluble form, such as a pharmaceutically
acceptable salt, which is meant to include both acid and base
addition salts. The composition may also include one or more of the
following: carrier proteins, such as serum albumin, buffers,
stabilizing agents, coloring agents, and the like. Additives are
well known in the art, and they are used in a variety of
formulations.
[0039] Furthermore, the
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine and derivatives
thereof, may be lyophilized and the resulting lyophilizates used,
for example, to produce preparations for injection. The basic
principles for obtaining lyophilizates are known to the skilled
artisan. A method for the production of lyophilizates with improved
dilution rate is exemplarily described in DE 199 03 275 A1, which
is incorporated as reference in the disclosure of the invention
hereby.
[0040] The preparations indicated may be sterilized and/or may
comprise auxiliaries, such as lubricants, preservatives,
stabilizers, fillers, chelating agents, antioxidants, solvents,
bonding agents, suspending agents, wetting agents, emulsifiers,
salts (for influencing the osmotic pressure), buffer substances,
colorants, flavorings and one or more further active substances,
for example one or more vitamins.
[0041] The concentration of the prophylactically or therapeutically
active ingredient in the formulation may vary from about 0.1 to 100
wt %. Preferably, the compound
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine or the
derivatives thereof are administered in doses of approximately 1 to
600 mg, more preferably between 5 and 100 mg per dose unit.
Generally, such a dose range is appropriate for total daily
incorporation. In other terms, the daily dose is between 0.02 and
200 mg/kg of body weight, preferably between 20 and 100 mg/kg of
body weight, more preferably between approximately 0.02 and 10
mg/kg of body weight. The specific dose for each patient depends,
however, on a wide variety of factors as already described in the
present specification.
[0042] The invention also relates to the use of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, and/or a
physiologically acceptable salt and/or solvate thereof, for the
production of a medicament for the prophylactic or therapeutic
treatment and/or monitoring of diseases that are associated with
insulin resistance and/or .beta.-cell dysfunction.
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, and/or a
physiologically acceptable salt and/or solvate thereof, can
furthermore be employed as intermediate for the preparation of
further medicament active ingredients. The medicament is preferably
prepared in a non-chemical manner, e.g. by combining the active
ingredient with at least one solid, fluid and/or semi-fluid carrier
or excipient, and optionally in conjunction with a single or more
other active substances in an appropriate dosage form.
[0043] The medicament can be used to prevent the initiation of
diseases associated with insulin resistance and/or .beta.-cell
dysfunction in advance or to treat the arising and continuing
symptoms. The diseases as concerned by the invention are preferably
T2DM and/or related diseases thereof, the latter are more
preferably selected from the group of the Metabolic syndrome,
diabetic nephropathy and neuropathy. The prior teaching of the
present specification concerning the pharmaceutical composition is
valid and applicable without restrictions to the use of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine and its
derivatives for the production of a medicament for prophylaxis and
therapy of said diseases.
[0044] The aforementioned medical products of the inventive use are
particularly used for the therapeutic treatment. Monitoring is
considered as a kind of treatment provided that the compound is
administered in distinct intervals, e.g. in order to booster the
response and eradicate the symptoms of the disease completely.
Either the identical compound or different compounds can be
applied. In the meaning of the invention, prophylactic treatment is
advisable if the subject possesses any preconditions for the onset
of T2DM, such as a familial disposition, a genetic defect, or a
previously passed disease.
[0045] Object of the present invention is also the use of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, and/or a
physiologically acceptable salt and/or solvate thereof, for the
enhancement of insulin sensitivity and/or the preservation or
increase of .beta.-cell compensation. Herein, preservation refers
to a similar value within a normal statistical range that is caused
by the measurement method and the fact of a living organism
involved. A standard deviation of maximal 10% shall be regarded as
preservation, preferably maximal 3% only. Contrary to that, the
insulin sensitivity will strongly exceed initial values. The
insulin sensitivity is at least doubled, preferably at least
tripled, more preferably at least quadrupled, and most preferably
at least quintupled.
[0046] The use according to the previous paragraph of the
specification may be either performed in-vitro or in-vivo models.
Their .beta.-cells are either susceptible to deterioration
themselves, i.e. they naturally loss function or undergo apoptosis,
respectively, or exposed to age-promoting substances, such as
pro-apoptotic substances. Similarly, somatic cells can be either
insulin resistant themselves, i.e. normal amounts of insulin are
inadequate to produce a normal insulin response, or exposed to
IR-promoting drugs, such as cortisone, TNF-alpha, PAI-1 or
resistin. Both, the aging processes, which are prevented, and the
insulin response, which is sensitized, can be monitored by the
techniques described in the course of the present specification.
The in-vitro use is preferably applied to samples of humans
suffering from T2DM. Testing of several specific derivatives of the
compound 2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine makes
the selection of that active ingredient possible that is best
suited for the treatment of the human subject. The in-vivo dose
rate of the chosen derivative is advantageously pre-adjusted to the
deterioration susceptibility and/or severity of IR of the
respective specific cells with regard to the in-vitro data.
Therefore, the therapeutic efficacy is remarkably enhanced.
Moreover, the prior teaching of the present specification
concerning the use of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine and its
derivatives for the production of a medicament for the prophylactic
or therapeutic treatment and/or monitoring is considered as valid
and applicable without restrictions to the use of the compound for
the prevention of reduced .beta.-cell compensation and increase of
IR if expedient.
[0047] It is another object of the invention to provide a method
for treating Type II diabetes mellitus, the Metabolic syndrome,
nephropathy and/or neuropathy, wherein an effective amount of
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine, and/or a
physiologically acceptable salt and/or solvate thereof, is
administered to a mammal in need of such treatment. The mammals to
be treated are humans in particular. The preferred treatment is an
oral or parenteral administration. The treatment of the patients
with T2DM or people bearing a risk of developing T2DM on the basis
of existing IR by means of the NHE1-inhibitor
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine improves the
whole-body insulin sensitivity and ameliorates IR in these
individuals. The prior teaching of the invention and its
embodiments is valid and applicable without restrictions to the
method of treatment if expedient.
[0048] In the scope of the present invention,
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine is used for the
prophylactic or therapeutic treatment and/or monitoring of human
diseases that are associated with insulin resistance and/or
.beta.-cell dysfunction for the first time. The invention addresses
the role of .beta.-cell compensation in response to prolonged
insulin resistance. The aforementioned effects are interrelated
such that 2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine acts
either as a peripheral insulin sensitizer (secondarily preserving
.beta.-cell function by reducing the secretory burden on the
pancreas) or as a direct insulin secretagogue (secondarily
improving insulin sensitivity by increasing tissue insulinization),
or both. As result of providing the pharmaceutical composition
according to the invention, the insulin sensitivity is increased
while the age-related decrease in .beta.-cell function
(compensation) is prevented, but may even be reversed. Its use is a
promising, novel approach for a broad spectrum of therapies causing
a direct and immediate reduction of symptoms. The impact is of
special benefit to efficiently combat T2DM and illnesses arising
from T2DM. The compound and derivatives thereof are characterized
by a high specificity and stability; low manufacturing costs and
convenient handling. These features form the basis for a
reproducible action, wherein the lack of cross-reactivity and
adverse effects is included, and for a reliable and safe
interaction with their matching target structures.
[0049] It is to be understood that this invention is not limited to
the particular pharmaceutical composition, use and method described
herein, as such matter may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to limit
the scope of the present invention, which is only defined by the
appended claims. As used herein, including the appended claims,
singular forms of words such as "a," "an," and "the" include their
corresponding plural referents unless the context clearly dictates
otherwise. Thus, e.g., reference to "an active ingredient" includes
a single or several different active ingredients, and reference to
"a method" includes reference to equivalent steps and methods known
to a person of ordinary skill in the art, and so forth. Unless
otherwise defined, all technical and scientific terms used herein
have the same meaning as commonly understood by a person of
ordinary skill in the art to which this invention belongs.
[0050] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable examples are described below. The
following examples are provided by way of illustration and not by
way of limitation. Within the examples, standard reagents and
buffers that are free from contaminating activities (whenever
practical) are used.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 shows the body weight of ZF (fa/fa) rats at the age
of 8-12 week during treatment (Mean+/-SD, n=12).
[0052] FIG. 2 shows the random morning glucose level of ZF (fa/fa)
rats at the age of 8-12 week during treatment (Mean+/-SD, n=12,
*p<0.05). Week 4 data are 4 hour fasted glucose readings from
GDT group.
[0053] FIG. 3 shows the fasting blood glucose level in ZF (fa/fa)
rats at the age of 12 weeks following 4 weeks of treatment
(Mean+/-SD, n=6).
[0054] FIG. 4 shows the fasting insulin level in ZF (fa/fa) rats at
the age of 12 weeks following 4 weeks of treatment (Mean+/-SD, n=6,
*p<0.01).
[0055] FIG. 5 shows the insulin responses (insulin AUC) to an oral
glucose load in ZF (fa/fa) rats at the age of 12 weeks following 4
weeks of treatment (Mean+/-SD, n=6, GDT, *p<0.01 vs.
pre-treatment).
[0056] FIG. 6 shows the glucose response (glucose AUC) to an oral
glucose load in ZF (fa/fa) rats at the age of 12 weeks following 4
weeks of treatment (Mean+/-SD, n=6, CDT).
[0057] FIG. 7 shows the pancreatic compensation as quantified by
deuterated glucose disposal test (.sup.2H-GDT; 90 minute % D.sub.2O
recovery) in ZF (fa/fa) rats at the age of 12 weeks following 4
weeks of treatment (Mean+/-SD, n=6, GDT group, *p<0.01).
[0058] FIG. 8 shows the insulin sensitivity as quantified by
deuterated glucose disposal test (.sup.2H-GDT; % D.sub.2O
recovery/insulin AUC) in ZF (fa/fa) rats at the age of 12 weeks
following 4 weeks of treatment (Mean+/-SD, n=6, *p<0.01).
[0059] FIG. 9 shows the body weights of Zucker fa/fa and lean
control rats during drug treatment period. Data are the
average+/-standard deviation; n=6/group (error bars are obscured by
symbols); *p<0.01 from control fa/fa rats.
[0060] FIG. 10 shows the random blood glucose determined once per
week at approximately 9 a.m. There were no significant differences
between groups. Data are the average+/-standard deviation;
n=6/group.
[0061] FIG. 11 shows the 4 h fasting blood glucose concentrations
as determined by a glucometer reading during 4 weeks of drug
treatment. Data are the average+/-standard deviation; n=6/group; #
different than lean control (Bonferroni posttest).
[0062] FIG. 12 shows the insulin concentrations after a 4 hour fast
determined at one week intervals. Data are the average+/-standard
deviation; n=6/group; * significantly different than fa/fa chow
(vehicle) control.
[0063] FIG. 13 relates to pretreatment and shows the blood glucose
and insulin concentrations in response to a glucose challenge
during the GDT prior to starting drug treatment. Data are the
average+/-standard deviation (error bars are obscured by symbol for
some groups); n 6 for Compound A 80 mg/kg and lean control; n=5 for
fa/fa veh, Compound A 40 mg/kg, Compound B 40 mg/kg; n=4 for
Compound B 80 mg/kg; * different than fa/fa vehicle control; #
significantly different than lean vehicle. FIG. 13a shows the
concentration at successive time points in response to a glucose
challenge. FIG. 13b shows the area under curve (AUC 60 min) for
glucose response graphed in FIG. 13a. FIG. 13c shows the
concentration at successive time points in response to a glucose
challenge. FIG. 13d shows the AUC 60 min insulin response graphed
in FIG. 13c. For FIGS. 13b and 13d the different groups are
represented by the bars from left to right: Lean Veh, fa/fa-Veh,
Cmp A 40 mpk, Cmp A 80 mpk, Cmp B 40 mpk, Cmp 80 mpk.
[0064] FIG. 14 relates to 2 weeks of drug treatment and shows the
blood glucose and insulin concentrations in response to a glucose
challenge during the GDT 2 weeks after starting drug treatment.
Data are the average+/-standard deviation (error bars are obscured
by symbol some groups); n=6 for fa/fa veh, Compound A 40 mg/kg,
Compound A 80 mg/kg, Compound B 40 mg/kg and lean control; n=4 for
Compound B 80 mg/kg; * different than fa/fa chow vehicle; #
significantly different than lean vehicle. FIG. 14a shows the
concentration of glucose at successive time points in response to
glucose challenge. FIG. 14b shows the area under curve (AUC 60 min)
for glucose response graphed in FIG. 14a. FIG. 14c shows the
concentration of insulin at successive time points in response to a
glucose challenge. FIG. 14d shows the AUC 60 min of the insulin
response graphed in FIG. 14c. For FIGS. 14b and 14d the different
groups are represented by the bars from left to right: Lean Veh,
fa/fa-Veh, Cmp A 40 mpk, Cmp A 80 mpk, Cmp B 40 mpk, Cmp 80
mpk.
[0065] FIG. 15 relates to 4 weeks of drug treatment and shows the
blood glucose and insulin concentration in response to a glucose
challenge during the 3.sup.rd GDT that occurred 4 weeks after
starting drug treatment. Data are the average+/-standard deviation
(error bars are obscured by symbols in some cases); n=6 for fa/fa
chow, Compound A 40 mg/kg, Compound B 40 mg/kg and lean control;
n=5 for Compound A 80 mg/kg, Compound B 80 mg/kg; * different than
fa/fa chow vehicle; # different than lean control. FIG. 15a shows
the concentration of glucose in response to a glucose challenge.
FIG. 15b shows the area under curve (AUC 60 min) for glucose
response graphed in FIG. 15a. FIG. 15c the concentration of insulin
at successive time points in response to a glucose challenge. FIG.
15d shows the AUC 60 min for insulin response graphed in FIG. 15c.
For FIGS. 15b and 15d the different groups are represented by the
bars from left to right: Lean Veh, fa/fa-Veh, Cmp A 40 mpk, Cmp A
80 mpk, Cmp B 40 mpk, Cmp 80 mpk.
[0066] FIG. 16 shows the insulin sensitivity (a, c, e) and
pancreatic compensation (b, d, e) in response to a glucose
challenge pretreatment (a, b), 2 weeks after start of treatment (c,
d) and 4 weeks after start of treatment (e, f). * significantly
different than vehicle treated (chow) fa/fa; # significantly
different than lean control (ANOVA followed by Tukey test). For
FIGS. 16a-16f the different groups are represented by the bars from
left to right: Lean Veh, fa/fa-Veh, Cmp A 40 mpk, Cmp A 80 mpk, Cmp
B 40 mpk, Cmp 80 mpk.
PHARMACOLOGICAL STUDY REPORT I
Study Design
[0067] Obese, insulin resistant ZF (fa/fa) rats, which are treated
from the age of 8-12 weeks, were used as IR model. Rats were housed
with a 12-h light/dark cycle and free access to food and water
unless otherwise noted. Studies were approved by the Institutional
Animal Care and Use Committee. There were a total of 18 animals in
the study (8 week-old males, provided by Charles River). Animals
were randomized into 3 groups (vehicle group, Compound A group,
Compound B group, n=6 per group) based on random glucose level at
the beginning of the study: [0068] Group 1: ZF rats (fa/fa) (n=6),
compound A (150 mg/kg via gavage) [0069] Group 2: ZF rats (fa/fa)
(n=6), compound B (40 mg/kg/day as admixture to the chow) [0070]
Group 3: ZF rats (fa/fa) (n=6), vehicle treatment (control chow,
vehicle gavage)
[0071] Group 1 and 3 animals were treated with compound A or
vehicle (water) for 4 weeks (from age 8-12 weeks) via oral gavage,
twice daily. Chow containing compound B was fed to Group 2 ad lib.
Group 1 and Group 3 were fed with chow without compound B ad
lib.
[0072] Compound A corresponds to Cariporide. Compound B corresponds
to 2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine hydrochloride
hydrate.
Blood Sampling and Plasma Glucose and Insulin Assays
[0073] Random morning blood was withdrawn from ad libitium fed rats
by a small cut in the tip of the tail. Blood was collected in
heparinized tubes. Blood glucose concentrations were measured as
per study design using a One-Touch glucometer (Lifescan Inc,
Milptias, Calif.). Blood samples were placed on ice and
centrifuged, and plasma stored at -20.degree. C. until insulin was
assayed. Rat plasma insulin concentration was measured with
rat-specific ELISA kit (Crystal Chem Inc, Downers Grove, Ill.).
.sup.2H-GDT
[0074] Rats underwent a 4 hour fast. One hour prior to the
.sup.2H-GDT challenge rats received 1.75 mg/g of body weight of
H.sub.2O.sup.18 (10% .sup.18O, Spectra, X XX) by oral gavage to
allow the determination of total body water as previously
described. At t=0, the blood glucose concentrations were determined
with a glucometer, and a blood sample was obtained for
determination of base line insulin and H.sub.2O.sup.18 dilution.
Animals then received [6,6'-.sup.2H]glucose (2 g/kg body weight,
50% in water, Cambridge Isotope Laboratory, Inc., Andover, Mass.)
by oral gavage. The 2 g/kg oral glucose load used in the rat
protocol achieved glucose levels within or near the desired dynamic
range. A second blood sample was collected at t=30 min for
determination of glucose and insulin concentrations and
.sup.2H.sub.2O content.
Heavy Water Labeling Protocol
[0075] Animals received an IP bolus (0.35 .mu.l/g body weight) of
99% heavy water in 0.9% NaCl to reach a body water enrichment of
roughly 5% (using an estimated 60% body weight as water) and then
received 8% .sup.2H.sub.2O in drinking water for last 4 weeks of
study. Body water .sup.2H.sub.2O enrichments reach stable
steady-state values within a few days in rodents on this
protocol.
IRMS Analysis
[0076] One hundred micro liter aliquots of plasma samples inside
the cape of an inverted vial were placed in a heating blocked
filled with glass beads at 70.degree. C. overnight and the water
distillate inside the vial was collected. The deuterium and
oxygen-18 isotope ratios of the blood of plasma samples were
determined using a Thermo Finnigan High Temperature
Conversion/Elemental Analyzer coupled with a Thermo Finnigan MAT
253 IRMS via a Conflo-III Interface. The first two measurements
were discarded to minimize hysteresis effects from the previous
sample. The deuterium isotope abundance is first calculated in
.delta. 2H values relative to the international VSMOW standard, and
then transformed to APE by using a calibration curve of standards
with known enrichments. The .sup.2H.sub.2O enrichment was
calculated for rats at 30 min after the glucose load.
.sup.2H.sub.2O enrichment was converted to mmoles by multiplying
enrichment by the TBW pool size and dividing by 20 (MW
.sup.2H.sub.2O). Total .sup.2H.sub.2O produced was calculated as a
percent of the [6,6'-.sup.2H.sub.2]glucose load given. Plasma
insulin (INS AUC) and glucose (GLU AUC) areas under the curves were
calculated using the trapezoidal method. Two .sup.2H-GDT parameters
were calculated: 1).sup.2H.sub.2O production (% load)/INS AUC*GLU
AUC and 2) the absolute rate of .sup.2H.sub.2O production (%
load).
Results
Body Weight
[0077] There were no significant differences in body weight (FIG.
1) among the vehicle-, Compound A- or Compound B-treated
groups.
Random Morning Blood Glucose Level
[0078] Random morning blood glucose levels were measured weekly
between 9-11 a.m. There was no significant difference in random
morning blood glucose levels between Compound B-treated and vehicle
groups during the 4 weeks of this study (FIG. 2). However, Compound
A-treated group trended towards lower glucose level by the end of
the study compared to the other two groups.
Fasting Glucose and Insulin Levels
[0079] At the end of the study, rats were fasted for 4 hours and
glucose and insulin level were measured. Fasting glucose levels in
Compound A- and Compound B-treated animals trended towards lower
values post treatment, while there were no significant differences
in the vehicle treated group, pre- and post-treatment (FIG. 3).
There were significantly lower fasting insulin levels in both
compound A- and Compound B-treated animals compared to their
pre-treatment levels (FIG. 4).
GDT
[0080] All animals underwent .sup.2H-GDT both prior and following
the 4 weeks treatment. For the .sup.2H-GDT, animals were fasted for
4 hours. One hour prior to the .sup.2H-GDT challenge, the rats
received 1.75 mg/g body weight of H.sub.2.sup.18O by oral gavage to
allow the determination of total water content. At t=0 min, a blood
sample was obtained for the determination of base line insulin,
glucose and H.sub.2.sup.18O dilution. Then,
[6,6'-.sup.2H.sub.2]glucose (2 g/kg body weight, 50% in water) was
administered to the animals by oral gavage. Serial blood samples
were taken for glucose and insulin concentrations and
.sup.2H.sub.2O content at 90 min time point. The adequacy of
pancreatic compensation was assessed by distinguishing between the
absolute rate of glucose utilization achieved (reflecting
pancreatic compensation to IR) and the glycolytic disposal of
administered glucose per unit of ambient insulin (reflecting
insulin sensitivity). The .beta.-cell compensation is expressed as
the % .sup.2H.sub.2O recovery after the oral
[6,6'-.sup.2H.sub.2]glucose load and the insulin sensitivity is
expressed as the % .sup.2H.sub.2O recovery per insulin AUC.
[0081] There were no significant differences in insulin AUC (FIG.
5) or glucose AUC levels (FIG. 6) among the three groups either
pre- or post-treatment. Glucose AUC during the oral glucose load
followed a similar trend as fasting glucose levels. As can be seen,
there was a large standard deviation, likely representing
physiologic variation. However, all post-treatment insulin AUC
levels were significantly lower compared to pre-treatment. The fall
in vehicle-treated animals likely represents the natural history of
.beta.-cell deterioration with aging (from age of 8 to 12 weeks) in
ZF rats.
[0082] Since the change of insulin AUC in Compound A- and Compound
B-treated animals may be either caused by age-related deterioration
or reduced insulin needs due to improved insulin sensitivity, the
reason was figured out by GDT measurement. There were no
significant differences among groups in pancreatic compensation,
both pre- and post-treatment. Comparisons of compensation before
and after treatment within each group, however, revealed that both
vehicle and compound A groups exhibited lower pancreatic
compensation post-treatment (FIG. 7), most likely due to the
natural progression of .beta.-cell dysfunction in ZF rats. In
contrast, Compound B-treated ZF rats maintained the same degree of
pancreatic compensation as was present at baseline. These results
suggest preservation of .beta.-cell function in response to
Compound B treatment only.
[0083] Consistent with these findings, insulin sensitivity measured
by the GDT (FIG. 8) showed that Compound B-treated animals had
significantly increased insulin sensitivity (.about.4 fold)
compared to pre-treatment values while there was no significant
change in insulin sensitivity post-treatment in the vehicle- and
Compound A-treated groups compared to pre-treatment, although there
was a trend toward increased values in both groups. Thus,
2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine hydrochloride
hydrate as represented by Compound B significantly increased
insulin sensitivity and prevented the age-related reduction in
.beta.-cell compensation, indicating that the reduction in insulin
AUC post-treatment was physiologic in Compound B-treated animals.
These results also emphasized the need to characterize insulin
sensitivity when assessing insulin concentrations in models of
insulin resistance.
PHARMACOLOGICAL STUDY REPORT II
Study Design
[0084] In the previous .sup.2H-GDT study (Pharmacological Study
Report I), Compound B increased insulin sensitivity (SI) and either
increased or maintained the .beta.-cell responsiveness (pancreatic
compensation) in Zucker fa/fa rats. However in this study, the
baseline fasting insulin levels differed between groups. There was
an overall reduction in fasting insulin levels and the insulin AUC
in all groups of animals, despite the fact that there were no
changes in random glucose, fasting glucose, or the glucose AUC in
any of the treatment groups. The objective of the present study was
to confirm the original observations of the effects of compounds A
and B on SI and pancreatic compensation (PC). A secondary objective
was to better define the time course of the drugs' effects on the
glucose and insulin responses to an oral glucose load. A number of
changes were made from the previous study: [0085] 1. Compounds A
and B were mixed with food to eliminate the stress of daily oral
gavage. The presence of drug in the diet did not adversely affect
food intake and would give an acceptable concentration of drug in
the plasma. [0086] 2. Zucker fa/fa rats were assigned to a
treatment group on the basis of preliminary measurements of body
weight, fasting glucose and insulin concentrations, and
glycosylated hemoglobin A1c levels (Gly-HbA1c), to eliminate
potential bias in the final results. [0087] 3. Fasting glucose and
insulin concentrations were determined weekly. [0088] 4.
.sup.2H-GDT was carried out on Days 0, 14 and 28. In addition,
during the .sup.2H-GDT, blood glucose and insulin levels were
determined at 0, 15, 30, 60 and 90 min to better define both the
insulin and glucose responses. [0089] 5. Glycosylated HbA1c levels
were determined on Days 0 and 28 to provide an additional
assessment of any overall improvement in glucose homeostasis
achieved by treatment. [0090] 6. A lean control group was included
in the study.
Subjects
[0091] Thirty-six obese Zucker fa/fa and six lean Zucker fa/? male
rats obtained from Charles River, were housed under normal vivarium
conditions on a 12 hour light/dark cycle. The rats were kept two to
a cage with food and water available ad lib, except during the
weekly, 4 hour fasts. Water continued to be available during these
fasts.
Drug
[0092] Compound A corresponds to Cariporide. Compound B corresponds
to 2-Methyl-4,5-di-(methylsulfonyl)-benzoyl-guanidine hydrochloride
hydrate. Compounds A and B were supplied in powder form by Merck
Serono. Drugs were incorporated into a pelleted diet (LabDiet,
5001) by Research Diets, Inc (New Brunswick, N.J.) at
concentrations of 0.44 g/kg or 0.88 g/kg of diet to yield
approximate doses of 40 or 80 mg/kg. The concentration was
calculated based on the assumption that a 300 g rat would eat about
30 g/d of chow. The same chow, but without drug, was fed to rats of
both the lean and fa/fa control group animals.
Group Assignment
[0093] One week prior to the start of the study, all rats were
weighed. After a 4 hour fast, a blood sample was obtained for the
determination of blood glucose, insulin, and glycosylated
hemoglobin levels (HbA1c). Animals were then sorted on the basis of
these parameters into the groups listed below. Rats were eight
weeks of age at the onset of treatment.
Treatment Groups:
[0094] 1. ZF rats (fa/fa) (n=6) chow without drug [0095] 2. ZF rats
(fa/fa) (n=6) compound A (40 mg/kg/day) [0096] 3. ZF rats (fa/fa)
(n=6) compound A (80 mg/kg/day) [0097] 4. ZF rats (fa/fa) (n=6)
compound B (40 mg/kg/day) [0098] 5. ZF rats (fa/fa) (n=6) compound
B (80 mg/kg/day) [0099] 6. lean Zucker rats (fa/?) (n=6) chow
without drug
Dosing Regime
[0100] Treatment appropriate chow was continuously available for 4
weeks except during scheduled 4 h fasts conducted once per
week.
Body Weight and Food Intake
[0101] individual body weights were determined once per week.
Average food intake per cage was also determined once per week and
the individual rat consumption was calculated by dividing the cage
average by 2 (rats housed 2/cage).
Glucose and Insulin Levels
[0102] Both random and fasting (4 h) blood glucose levels were
determined using a hand-held glucometer (OneTouch Ultra, Lifescan,
Inc. Milapitas, Calif.) on blood obtained from tail capillary
samples. Plasma insulin levels were determined by a rat specific
ELISA (Ultra Sensitive Rat Insulin ELISA Kit, Crystal Chem, Inc.,
Chicago, Ill.). The 4 h fast period started at 9 a.m. and ended
after a blood draw at 1 p.m.
HbA1c Levels
[0103] HbA1c level were determined using a DCA 2000 analyzer (Bayer
Healthcare LLC, Elkhart Ind.) prior to beginning the study and
again at the end of the 4.sup.th week treatment period.
.sup.2H-GDT
[0104] Insulin sensitivity (SI) and pancreatic compensation (PC)
were measured by conducting .sup.2H-Glucose disposal test on Days
0, 14 and 28.
[0105] Time -4 h: Before the fast was begun on the morning of the
GDT, a blood sample was taken to determine the random glucose level
(see above). An additional blood sample was collected to determine
the baseline .sup.2H.sub.2O and .sup.18O enrichments. The rats were
weighed and the 4 h fast was begun. Water was available during the
fast.
[0106] Time -3 h: One 1 hour after the start of the fast, 1.75 mg/g
body weight of 10% H.sub.2.sup.18O was administered by oral gavage
(.sup.2H.sub.2O-free H.sub.2O.sup.18 water; 10% .sup.18O atom from
Spectra; Cat. No 51350).
[0107] Time 0: 4 hours after the start of the fast, the blood
glucose and insulin levels were determined. Rats received 4 ml/kg
of a 50% glucose solution (comprised of 25% [6,6-.sup.2H.sub.2]
glucose and 25% D-glucose) by oral gavage.
[0108] At times 15, 30, 60 and 90 min post glucose challenge, blood
glucose concentrations were again determined by a glucometer
reading from tail vein blood. Additional blood samples were
collected for insulin determinations. At 60 and 90 min post glucose
challenge, whole blood was collected to determine the
.sup.2H.sub.2O and H.sub.2.sup.18O enrichments. All blood samples
were centrifuged and the plasma was stored at -20.degree. C. until
assayed. The deuterium and oxygen-18 isotope enrichments of body
water were determined by IRIS analysis of the water distillate of
the plasma samples.
Calculations
[0109] .sup.2H.sub.2O enrichment was converted to mmol by
multiplying enrichment (.sup.2H.sub.2O APE) by the total body water
pool size and dividing by 20 (molecular weight of .sup.2H.sub.2O).
Total .sup.2H.sub.2O produced was calculated as the percent of the
[6,6-.sup.2H.sub.2] glucose load administered. Plasma insulin and
glucose areas under the curve (AUC 60 min) were calculated using
the trapezoidal method (GraphPad Prism).
[0110] Two parameters were calculated from the .sup.2H-GDT: [0111]
1. Insulin sensitivity index (SI)=.sup.2H.sub.2O production (%
load)/(glucose AUC.times.insulin AUC). [0112] 2. Pancreatic
compensation (PC)=the absolute .sup.2H.sub.2O production as a
percent of total [6,6-.sup.2H.sub.2] glucose load (% load)/the
integrated glucose response (glucose AUC; to correct for
insulin-independent utilization).
Statistics
[0113] Overall results of glucose and insulin measures were
analyzed by a 2 way repeated measures ANOVA using drug and time
(repeated) as factors. The primary analysis was followed by
Bonferroni posttests to compare all groups. Data are considered
significant at p<0.05. AUC 60 min values for glucose and insulin
were analyzed by 1 way ANOVA followed by Tukey's comparison of all
groups.
Data Exclusion
[0114] Data that indicated that there was a lack of enrichment due
to a failure, or partial failure of glucose administration during
the .sup.2H-GDT, were omitted.
Results
Pre-Study Group Assignment
[0115] There were no significant differences between fa/fa groups
at the start of the study. As expected, the lean animals' body
weight and fasting insulin levels differed significantly from all
fa/fa animals. Although the glycosylated hemoglobin and fasting
glucose levels were somewhat lower in lean animals, these values
were not significantly different from those of other treatment
groups (Table 1).
TABLE-US-00001 TABLE 1 Pre-study body weights, fasting blood
glucose, insulin and Gly-HbA1c levels. Data are the Means .+-. SEM,
n = 6/group; *p < 0.001, differs significantly compared to all
fa/fa treatment groups. Lean fa/? fa/fa fa/fa cmp A fa/fa cmp A
fa/fa cmp B fa/fa cmp B Group Chow chow 40 mg/kg 80 mg/kg 40 mg/kg
80 mg/kg BW 259.6 .+-. 2.7* 378.9 .+-. 12.6 381.2 .+-. 15.3 377.2
.+-. 12.6 379.1 .+-. 13.0 376.3 .+-. 11.6 (g) BG 100.6 .+-. 2.6
113.6 .+-. 6.0 113.2 .+-. 6.4 118.7 .+-. 10.3 109.5 .+-. 6.7 122.8
.+-. 6.4 (mg/dl) Insulin 1.0 .+-. 0.2* 10.5 .+-. 1.4 10.3 .+-. 1.0
10.8 .+-. 1.7 11.1 .+-. 0.8 10.3 .+-. 0.6 (ng/ml) HbA1c 3.5 .+-.
0.1 3.8 .+-. 0.1 4.0 .+-. 0.2 3.9 .+-. 0.1 3.8 .+-. 0.1 4.0 .+-.
0.1 (%)
Body Weight and Food Intake
[0116] All animals continued to gain weight for the duration of the
experiment, indicating that the drugs were well tolerated (2 way
repeated measure ANOVA followed by Bonferroni posttests: time:
F.sub.(4,136)=1488.41, p<0.0001; treatment: F.sub.(6,36)=31.02,
p<0.0001, interaction treatment x time: F.sub.(24,138)=12.69,
p<0.0001). No significant difference in body weight was noted
between the fa/fa groups over the 4 week treatment period (FIG. 9),
although all were increased relative to the lean control group. The
average weekly food intake of the Compound A and Compound B groups
was increased compared to lean controls, but did not differ
significantly from that of chow fed fa/fa controls (Table 2).
TABLE-US-00002 TABLE 2 Average food consumption in grams during the
4 week treatment period. Data are the average +/- standard
deviation; *p < 0.01 from chow fa/fa control. Treatment Week 1
Week 2 Week 3 Week 4 Chow fa/fa control 34.1 +/- 2.0 32.7 +/- 5.6
34.7 +/- 1.5 35.3 +/- 1.9 Compd A 40 mg/kg 32.2 +/- 3.5 33.1 +/-
2.6 33.9 +/- 2.4 34.9 +/- 2.7 Compd A 80 mg/kg 34.3 +/- 2.5 32.8
+/- 4.3 32.3 +/- 4.5 30.8 +/- 5.6 Compd B 40 mg/kg 32.1 +/- 3.2
33.8 +/- 2.9 31.7 +/- 2.9 33.6 +/- 4.4 Compd B 80 mg/kg 31.8 +/-
1.4 32.9 +/- 1.9 33.0 +/- 0.7 34.0 +/- 2.9 Lean control 21.2 +/-
1.5* 22.0 +/- 1.3* 21.1 +/- 0.8* 21.8 +/- 0.9*
Random Blood Glucose
[0117] Random blood glucose was determined once per week at
approximately 9 a.m. in non-fasted rats. There was no significant
difference between the random glucose levels of the chow fa/fa
group and any of the other treatment groups (FIG. 10) (2 way
repeated measure ANOVA followed by Bonferroni posttests: treatment
F.sub.6,136=0.93; p=0.48 ns; time: F.sub.4,136=3.25, p=0.01;
interaction F.sub.24,136=0.97, p=0.51 ns).
Fasting Blood Glucose Levels
[0118] Fasting blood glucose was determined once per week after a 4
h fast (fast at 9 a.m., sample at 1 p.m.). Glucose levels in the
various drug treatment groups did not differ significantly from the
control fa/fa group (FIG. 11). The 40 mg/kg dose of Compound A
increased the fasting glucose concentration after 4 weeks of
administration (2 way repeated measure ANOVA followed by Bonferroni
posttests: treatment: F.sub.6,136=5.26; p=0.0006; time:
F.sub.4,136=7.68, p=0.0001; interaction F.sub.24,136=0.97, p=0.51
ns).
Fasting Insulin Concentrations
[0119] Fasting insulin concentrations at baseline and after 4 weeks
of treatment are summarized in Table 3 (2 way repeated measure
ANOVA followed by Bonferroni posttests: treatment:
F.sub.6,36=11.24, p<0.0001; time: F.sub.6,36=9.48, p<0.0001;
interaction: F.sub.24,136=3.21, p<0.0001). The insulin
concentrations of the lean controls were significantly different
from those of the chow fed fa/fa control at each week, with the
exception of the 3.sup.rd week. By the 4.sup.th week of treatment
both Compound A at 80 mg/kg significantly decreased fasting insulin
relative to the fa/fa vehicle control. These values, however,
remained higher than those of the lean controls (FIG. 12).
Glycosylated Hemoglobin
[0120] HbA1c levels reflect glycemic control over time. At
baseline, the mean HbA1c levels of all fa/fa groups were somewhat,
but not significantly, higher than that of lean controls. At week
4, the HbA1c levels of the fa/fa control group and the Compound B
80 mg/kg group were significantly higher than those of the lean
controls (Table 3). The changes in HbA1c were not statistically
significant between any of the treatment groups over the 4 week
treatment period (Table 3).
TABLE-US-00003 TABLE 3 Baseline, Week 4 and the change over the 4
week treatment period of fasting insulin and HbA1c. Data are the
Means .+-. SD, n = 5-6/group; *significantly different than fa/fa
vehicle (chow) control; .sup.#significantly different than lean
control (Tukey test, p < 0.05). Lean fa/? fa/fa cmp A fa/fa cmp
A fa/fa cmp B fa/fa cmp B Group Chow Fa/fa chow 40 mg/kg 80 mg/kg
40 mg/kg 80 mg/kg Baseline 1.4 .+-. 0.5* 13.4 .+-. 2.9.sup.# 17.6
.+-. 5.1.sup.# 15.9 .+-. 5.5.sup.# 14.7 .+-. 2.7.sup.# 16.2 .+-.
11.2.sup.# Insulin (ng/ml) Week 4 Insulin 1.7 .+-. 0.5* 21.6 .+-.
10.7.sup.# 18.2 .+-. 11.8.sup.# 12.5 .+-. 7.5*.sup.# 14.5 .+-.
5.5.sup.# 13.1 .+-. 8.2.sup.# (ng/ml) Change in 0.3 .+-. 0.8 8.2
.+-. 9 0.6 .+-. 9.6 -3.4 .+-. 8.2 -0.2 .+-. 4.4 -3.2 .+-. 11.2
Insulin (ng/ml) Base line 3.5 .+-. 0.1 3.8 .+-. 0.4 4.0 .+-. 0.5
3.9 .+-. 0.4 3.8 .+-. 0.2 4.0 .+-. 0.4 HbA1c (%) Week 4 3.8 .+-.
0.1* 4.7 .+-. 0.7.sup.# 4.4 .+-. 0.2 4.4 .+-. 0.52 4.3 .+-. 0.3 4.8
.+-. 0.4.sup.# HbA1c (%) Change in 0.4 .+-. 0.2 0.9 .+-. 0.4 0.4
.+-. 0.6 0.4 .+-. 0.9 0.4 .+-. 0.2 0.8 .+-. 0.7 HbA1c (%)
.sup.2H-Glucose Disposal Test
[0121] To determine the effect of compounds A and B on pancreatic
function and insulin sensitivity, rats underwent a .sup.2H-GDT
prior to animals being weaned onto chow with drug, and then after 2
and 4 weeks of drug treatment
Pretreatment .sup.2H-GDT Glucose and Insulin Response
[0122] In response to a glucose challenge before the start of drug
treatment, blood glucose concentrations were increased in all fa/fa
rats relative to the lean controls (FIG. 13a). At the 60 min time
point, the glucose levels of both the 40 and 80 mg/kg Compound A
to-be-assigned groups were increased above those of the fa/fa
vehicle to-be-assigned animals. However, the glucose AUC (60 min)
indicated that all groups of fa/fa rats had a similar degree of
impaired oral glucose tolerance relative to the lean controls (FIG.
13b). At baseline, all fa/fa animals displayed a similar degree of
impaired hyperinsulinemia compared to the lean controls during the
.sup.2H-GDT challenge (FIG. 13c, d).
Two Week .sup.2H-GDT Glucose and Insulin Response
[0123] 2 weeks after the start of drug treatment, all fa/fa animals
had a significant increase in blood glucose concentrations relative
to the lean controls (FIG. 14a, b). Likewise all fa/fa animals had
increased insulin concentrations relative to the lean controls
(FIG. 14c, d). Compounds A and B did not affect the glucose or
insulin response after 2 weeks of treatment.
Four Weeks GDT Glucose and Insulin Response
[0124] 4 weeks after the start of drug treatment, rats dosed with
compound A (40 and 80 mg/kg) and compound B (40 and 80 mg/kg)
continued to have elevated blood glucose and insulin concentrations
(FIG. 15a, b, c, d). In fact at the 90 min time point, the glucose
level was elevated above that of the fa/fa vehicle treated rats in
the Compound A 40 mg/kg treated group. At 80 mg/kg, Compound B
slightly decreased blood glucose levels such that the values were
not significantly different from those of the lean vehicle rats or
the fa/fa vehicle controls. Insulin concentrations remained
elevated in the 80 mg/kg Compound B group.
Pancreatic Function and Insulin Sensitivity
Pretreatment
[0125] Insulin sensitivity and pancreatic compensation determined
at baseline were similar in all fa/fa groups (FIG. 16a, b) and were
significantly reduced compared to lean controls. This indicates the
ability of fa/fa rats to compensate to their severe insulin
resistance is incomplete and contributes to their glucose
intolerance. The apparent reduction in pancreatic compensation
observed in the group of animals that were later treated with 80
mg/kg of Compound B may be considered to be an experimental
artifact since this decrease was not observed at later time points
and these animals did not differ on other measures made at the same
time (e.g. weight, fasting glucose or insulin concentrations).
Results of ANOVA for PC: F.sub.6,29=33.02, p<0.0001; for SI:
F.sub.6,29=35.8, p<0.0001.
Two Weeks of Treatment
[0126] After 2 weeks of treatment, pancreatic compensation in the
Compound B, 80 mg/kg group was intermediate between that of the
lean and fa/fa control groups (FIG. 16c, d). Results of ANOVA for
PC: F.sub.6,32=9.121, p<0.0001; for SI F.sub.6,32=132.7,
p<0.0001.
Four Weeks of Treatment
[0127] After 4 weeks of drug treatment, the results were similar to
those of 2 weeks (FIG. 16e, f). Although insulin sensitivity in the
80 mg/kg Compound B group was not significantly improved, there was
a slight improvement in pancreatic compensation. Results of ANOVA
for PC: F.sub.6,32=6.17, p=0.0002; for SI: F.sub.6,32=35.01,
p<0.0001.
Conclusions
[0128] At baseline, there were no significant differences between
the fa/fa groups in body weight, food consumption, HbA1c, random
blood glucose, fasting blood glucose or insulin concentrations.
Likewise, in response to a glucose challenge, fa/fa groups did not
differ from one another in the glucose AUC (60 min) or insulin AUC
(60 min), but were significantly different than the lean controls.
Both insulin sensitivity and pancreatic compensation were similar
in all fa/fa rats at baseline, with the exception of the Compound B
(80 mg/kg/d) group which was depressed relative to the other
groups. After 4 weeks of treatment, 80 mg/kg of Compound A
decreased fasting insulin concentrations, but was without effect on
any of the other parameters. Neither dose of Compound A
significantly improved glucose levels (either fasting or in
response to challenge), insulin sensitivity or pancreatic
compensation. The effects of Compound B in the present experiments
were moderate. After 4 weeks of treatment at 80 mg/kg/d, Compound B
decreased glucose AUC 60 min in response to a glucose challenge.
The glucose concentration was intermediate between that of the lean
and fa/fa vehicle controls, but slightly different from either.
There was practically no effect on the insulin AUC 60 min, or on
the computed measure of insulin sensitivity (SI). Pancreatic
compensation appeared to be slightly improved after both 2 and 4
weeks of treatment and was slightly different from either the lean
or the fa/fa vehicle controls. It is noted, however, that
pancreatic compensation in this group appeared to be lower (worse)
than that of the other groups at baseline. 4 weeks of treatment
with Compound A did not improve glucose tolerance or significantly
improve the hyperinsulinemia and exaggerated insulin responses
observed in fa/fa rats. The high dose of Compound B attenuated the
deterioration in pancreatic compensation relative to the fa/fa
controls. Compound B has a positive effect on pancreatic function,
even though it does not effectively increase insulin
sensitivity.
Examples Related to Pharmaceutical Preparations
Example A: Injection Vials
[0129] A solution of 100 g of an active ingredient according to the
invention and 5 g of disodium hydrogen phosphate in 3 l of
bidistilled water is adjusted to pH 6.5 using 2 N hydrochloric
acid, sterile filtered, transferred into injection vials,
lyophilized under sterile conditions and sealed under sterile
conditions. Each injection vial contains 5 mg of active
ingredient.
Example B: Suppositories
[0130] A mixture of 20 g of an active ingredient according to the
invention is melted with 100 g of soya lecithin and 1400 g of cocoa
butter, poured into moulds and allowed to cool. Each suppository
contains 20 mg of active ingredient.
Example C: Solution
[0131] A solution is prepared from 1 g of an active ingredient
according to the invention, 9.38 g of NaH.sub.2PO.sub.4.2H.sub.2O,
28.48 g of Na.sub.2HPO.sub.4.12H.sub.2O and 0.1 g of benzalkonium
chloride in 940 ml of bidistilled water. The pH is adjusted to 6.8,
and the solution is made up to 1 l and sterilized by irradiation.
This solution can be used in the form of eye drops.
Example D: Ointment
[0132] 500 mg of an active ingredient according to the invention
are mixed with 99.5 g of Vaseline under aseptic conditions.
Example E: Tablets
[0133] A mixture of 1 kg of an active ingredient according to the
invention, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc
and 0.1 kg of magnesium stearate is pressed to give tablets in a
conventional manner in such a way that each tablet contains 10 mg
of active ingredient.
Example F: Coated Tablets
[0134] Tablets are pressed analogously to Example E and
subsequently coated in a conventional manner with a coating of
sucrose, potato starch, talc, tragacanth and dye.
Example G: Capsules
[0135] 2 kg of an active ingredient according to the invention are
introduced into hard gelatin capsules in a conventional manner in
such a way that each capsule contains 20 mg of the active
ingredient.
Example H: Ampoules
[0136] A solution of 1 kg of an active ingredient according to the
invention in 60 l of bidistilled water is sterile filtered,
transferred into ampoules, lyophilized under sterile conditions and
sealed under sterile conditions. Each ampoule contains 10 mg of
active ingredient.
Example I: Inhalation Spray
[0137] 14 g of an active ingredient according to the invention are
dissolved in 10 l of isotonic NaCl solution, and the solution is
transferred into commercially available spray containers with a
pump mechanism. The solution can be sprayed into the mouth or nose.
One spray shot (about 0.1 ml) corresponds to a dose of about 0.14
mg.
* * * * *